Reducing/Oxidizing Activity of Maternal Urine As Indicator of Fetal Gender Related Characteristics

ABSTRACT

The present invention provides a method for determining the gender of an unborn child by assaying the overall reducing/oxidizing or redox activity of the maternal urine or other body fluid. The method can be used to determine fetal gender at any time point during the entire pregnancy, the earliest being the first day of a missed menstruation. The body fluid may be processed before assaying. Processing may involve aging the body fluid, or purification of various fractions. The methods of the present invention also provide for a means for pre-conception baby gender planning by assaying the overall redox activity of a non-pregnant female&#39;s urine or other body fluid. The overall redox activity of a urine sample correlates with the gender specific compatibility of the ovum being released during a particular menstrual cycle. Therefore, assaying the overall redox activity of a non-pregnant female&#39;s urine will help a couple conceive a baby having a desired gender.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of the U.S.Provisional Application No. 61/069,008, filed on Mar. 11, 2008.

FIELD OF THE INVENTION

The present invention relates to a method for determining the gender ofan unborn child by evaluating the overall reducing/oxidizing activity ofthe maternal urine. The present invention also relates to a method forpre-conception baby gender planning by evaluating the overallreducing/oxidizing activity of a non-pregnant female's urine prior toconception.

BACKGROUND OF THE INVENTION

There is a great interest in accurately determining the gender of anunborn child as early as possible. Available techniques are typicallyperformed at late stages of a pregnancy. Ultrasound can be used todetermine fetal gender after the 18th week of pregnancy. Polymerasechain reaction (PCR) amplification of Y-specific DNA sequences requiresa sample of maternal blood, as well as expensive equipment. PCR can becarried out as early as the 6th week of gestation with an accuracy ofonly around 80%. Lagona et al. Multiple testing in fetal genderdetermination from maternal blood by polymerase chain reaction. Hum.Genet. 102, 6:1 (1998). Amniocentesis is performed after the 18th weekof pregnancy; however, it carries a risk of miscarriage due to itsinvasiveness. Chorionic Villi Sampling (CVS), conducted between the 10thand 13th week of pregnancy, can also provide accurate information, butagain, the procedure is invasive and requires costly equipment.

Alternatively, it is possible to determine the gender of an unborn childby assaying sex hormones. Sex hormones are steroids that play importantroles in both normal growth and development. In addition, sex hormonesinfluence the development of sex organs and maintenance of secondary sexcharacteristics in mammals. Testosterone, the principal male sexhormone, is primarily secreted by the testes of males and, to a muchless extent, the ovaries of females. On average, an adult man producesabout forty to sixty times more testosterone than an adult woman.Similarly, estrogen functions as the primary female sex hormone and isusually present at significantly higher levels in women than men. Theurine from a mother carrying a male fetus is richer in testosterone whencompared with the urine from a mother carrying a female fetus. Thisdifference is probably due to the contribution of sex hormones secretedby fetal testes. Jost A. A new Look at the Mechanisms Controlling SexDifferentiation in Mammals. John Hopkins Med. J. 130: 38 (1972).Approaches based on a pregnant woman's individual sex hormone levelshave been explored to determine fetal gender; however, thus far, theyhave not resulted in the development of any statistically reliablemethods. For example, in one study, antibodies against testosterone wereused in a radio-immunoassay to determine the testosterone levels inpregnant mothers' urine. The accuracy of this study with respect todetermining fetal gender was low. Loewit et al. Determination of fetalsex from maternal testosterone excretion in early pregnancy. Dtsch. Med.Wschr. 99: 1656 (1974).

Urine from pregnant women has been used in various attempts to developsimple and non-invasive tests to determine fetal gender. Most of theseprocedures exploit simple characteristics of maternal urine such as itspH or the ability to form complexes with aluminum compounds.Consequently, these tests are usually associated with comparatively poorreliability. For example, U.S. Pat. No. 6,420,182 discloses a method forfetal gender detection by assaying the pH of maternal urine after the12th to 14th week of pregnancy. The test is based on the hypothesis thatwomen bearing female fetuses have acidic urine, but the accuracy of thismethod is only about 65%. In another assay, a calorimetric test on urinefrom pregnant women may be performed after the 20th week of pregnancy.U.S. Pat. No. 4,840,914. The accuracy of this test in determining fetalgender was similarly low, only about 60% (see also,www.intelligender.com).

The applicant has noted that an evaluation of the reducing/oxidizingactivity of a urine sample from a pregnant female provides a bettermethod in determining fetal gender.

Depending upon the processing and assay methods, the hormones which cancontribute to the reducing activity of a urine sample are progesterone,testosterone and human chorionic gonadotropin (hCG), whereas estrogenscan contribute to the oxidizing activity of the urine. The primaryhormones directly linked to fetal gender are sex hormones, namely,testosterone and estrogens. The overall reducing/oxidizing activity ofthe urine sample from a pregnant mother carrying a male fetus isdifferent from the overall reducing/oxidizing activity of the urinesample from a pregnant mother carrying a female fetus allowing fordetermination of fetal gender.

There is a continuing need to develop a simple, non-invasive andreliable method for determining the gender of an unborn child as earlyas possible after conception. There is also a need for pre-conceptionbaby gender planning using a simple, non-invasive and reliabletechnique. Because human and other mammals have similar reproductivebiology, these methods may also be of great commercial value to animalbreeders.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordetermining the gender of an unborn child comprising the steps of: (a)contacting a body fluid from a pregnant female with at least one redoxindicator; (b) measuring redox activity of the body fluid; and, (c)comparing the redox activity of the body fluid with at least onestandard to determine the gender of the unborn child. The body fluid maybe selected from the group consisting of urine, tears, saliva, sweat,blood, plasma, serum, cerebrospinal fluid and amniotic fluid. The bodyfluid may be urine. The body fluid may be obtained during a periodranging from about the first day of a missed menstruation to about 40weeks of pregnancy. The body fluid may also be obtained during a periodranging from about 5 weeks to about 15 weeks of pregnancy.

The redox indicator may comprise a heteropoly acid or its salts, such asphospho-24-tungstic acid. The redox indicator may be a chromogenicchemical. The chromogenic chemical may comprise an oxidation-reductionsensitive metallic ion. The metallic ion may be selected from the groupconsisting of copper, iron, chromium, tungsten and molybdenum ions in anoxidized state. The chromogenic chemical may be selected from the groupconsisting of ferric tripyridyltriazine (Fe(III)-TPTZ) complex andpotassium ferricyanide. The redox activity may be measured using a freeradical, such as hydroxyl radical. Sodium salicylate may be used as theredox indicator. The redox activity may also be measured using anelectrochemical sensor. The redox activity may be measured on a solidsubstrate which comprises a redox indicator, such as a chromogenicchemical. The chromogenic chemical may be impregnated on a strip.

The body fluid may be processed prior to step (a). The body fluid may beaged for at least about 1 week at room temperature ranging from about20° C. to about 30° C. prior to step (a). The body fluid may be aged atroom temperature for a period of time ranging from about 1 week to 52weeks prior to step (a). The processing of the body fluid may comprisechemical, biochemical, physical or biological means, such as enzymatictreatment, extraction and purification. The processing may compriseusing adsorbants selected from the group consisting of talc,silica-based particles such as silica gel, alumina, florisil, charcoal,kaolin, concanavaline A and its conjugates, calcium phosphate, calciumhydroxide, calcium chloride, Cetyltrimethyl ammonium bromide, lectin,protein or glycoprotein hydrolyzing enzymes, glassfiber filter, ionexchange resins, affinity ligands, organic solvents, solid phaseextractants, size exclusion sieves, and reverse phase chromatographicmaterials. The processing may also comprise using precipitants, such asheavy metals selected from the group consisting of barium, lead,molybdenum and tungsten. The precipitants may also be selected from thegroup consisting of barium chloride, barium hydroxide, zinc chloride,mercuric chloride, lead acetate, ammonium sulphate, dextran,acetonitrile, chloroform, sodium hydroxide, trichloroacetic acid,potassium iodate and their mixtures.

The present invention also provides a kit comprising, (a) a urinecollecting vial; (b) a solid substrate comprising a redox indicator,wherein the redox indicator comprises at least one chromogenic chemical;(c) printed material instructing a woman to collect urine and contactthe urine with the solid substrate; and (d) printed material instructinga woman to compare redox activity of the urine sample with at least onestandard to determine the gender of the unborn child based on change incolor in the chromogenic chemical. The kit may further comprisechemicals to process urine. The printed material may further instruct awoman to process the urine prior to contact the urine with the solidsubstrate.

The present invention further provides a method for pre-conception babygender planning comprising the steps of: (a) contacting a body fluidfrom a non-pregnant female with at least one redox indicator; (b)measuring redox activity of the body fluid; and (c) comparing the redoxactivity of the body fluid with at least one standard to determinegender specific compatibility of the ovum released in a menstrual cycle.The body fluid may be urine. The body fluid may be obtained nearovulation or middle of the menstrual cycle. The body fluid may beprocessed prior to step (a).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining the gender of anunborn child by assaying the overall reducing/oxidizing or redoxactivity of the maternal urine.

The overall redox activity may be measured in other body fluids such astears and cerebrospinal fluid. The method can be used to determine fetalgender at any time point during the entire pregnancy, i.e., from thefirst day of a missed menstruation to about 40 weeks of pregnancy. Inorder to improve the accuracy of results, the body fluid may beprocessed before assaying. Processing may involve aging the body fluid,or purification of various fractions. For example, processing cancomprise aging the urine by storing it at ambient temperature (e.g.,approximately, 20-30° C.) for a defined period of time, e.g., 1-4 weeks,treating the urine by physical, chemical or biochemical means toaccelerate the aging process, and/or removing various urinary componentsby physical, chemical or biochemical means. In one embodiment, the bodyfluid is obtained from the pregnant female between the 5th and 15thweeks of pregnancy. Repeated analysis of urine samples obtained from thesame test subject at different time points in the pregnancy appears toimprove the accuracy of the analytical results. For example, if threeurine samples are analyzed, we have found that the accuracy of theresults approaches 100%.

The methods of the present invention also provide for a means forpre-conception baby gender planning by assaying the overall redoxactivity of a non-pregnant female's urine or other body fluid. The bodyfluid may be assayed at anytime during a menstrual cycle, but preferablyis assayed around the time of ovulation or middle of the menstrualcycle. Without being limited to any specific physiological mechanism, itis believed that ovulation generates at least two types of physiologicalconditions during alternate menstrual cycles, presumably due to therelease of two different types of ova. One type of ovum is compatiblewith fertilization by a Y chromosome-bearing sperm to produce a malefetus, whereas the other type of ovum is compatible with fertilizationby an X chromosome-bearing sperm to produce a female fetus. Thefertilization rate for a sperm and an incompatible ovum is very low. Wehave found that the overall redox activity of a urine sample correlateswith the gender specific compatibility of the ovum being released duringa particular menstrual cycle. Therefore, assaying the overall redoxactivity of a non-pregnant female's urine will help a couple conceive ababy having a desired gender.

The methods of the present invention may encompass assaying any suitablebody fluid, including urine, tears, saliva, sweat, blood, plasma, serum,cerebrospinal fluid and amniotic fluid. Pigman et al. The reducing powerof human saliva and its component secretions. J. Dent Res. 37(4):688-696 (1958). Oyawoye et al. Antioxidants and reactive oxygen speciesin follicular fluid of women undergoing IVF. Human Reproduction. 18(11): 2270-2274 (2003).

The term “overall redox activity” or “redox activity” of the body fluidrefers to the net redox activity (including antioxidant activity) of abody fluid as a whole, although various components of the body fluid mayexhibit different redox activities individually.

The overall redox activity of the body fluid such as urine depends notonly on the composition of the urine, but also, on how the sample isprocessed before assaying, as well as on the assay conditions. Forexample, the body fluid may be “aged” for at least about 1 week at roomtemperature ranging from about 20° C. to about 30° C. before assaying.The body fluid may also be aged for longer periods of time such as about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 months. The body fluid may beaged at room temperature for a period of time ranging from about 1 weekto about 52 weeks prior to assaying. Aging of the urine sample can rangefrom as long as 1 year to about 5 years, but longer periods may also beappropriate. Other suitable chemical, biochemical or physical treatmentsmay also be used to process the urine before assaying.

The term “fetal gender related characteristics” refers to (i) theevaluation of type and compatibility of ovum released in a particularmenstrual cycle before pregnancy, and (ii) the determination of sex ofthe fetus after pregnancy. The term “gender specific compatibility”refers to the compatibility of the ovum with fertilization by either a Ychromosome-bearing sperm to produce a male fetus, or by an Xchromosome-bearing sperm to produce a female fetus.

The present invention provides a method for determining the gender of anunborn child comprising the steps of: (a) contacting a body fluid from apregnant female with at least one redox indicator; (b) measuring redoxactivity of the body fluid; and, (c) comparing the redox activity of thebody fluid with at least one standard to determine the gender of theunborn child.

Because the ability of the body fluid to reduce or oxidize a reagentwill depend on the particular reagent (reducing agent or oxidizingagent) that is used to measure reduction or oxidation, the redoxactivity of the body fluid from a test subject is compared with theredox activity of a standard, i.e., the body fluid from a pregnantfemale where the sex of the fetus is known, to determine the gender ofthe fetus carried by the test subject. Alternatively, the redox activityof the body fluid from a test subject is compared with the redoxactivity of the pooled body fluid samples from pregnant females wherethe sexes of the fetuses are known, to determine the gender of the fetuscarried by the test subject. Similarly, for pre-conception methods, theredox activity of the body fluid from a test subject is compared withthe redox activity of a standard, i.e., body fluid from a non-pregnantfemale where the gender specific compatibility of the ovum the femalegenerated is known, to determine the gender specific compatibility ofthe ovum produced by the test subject. Alternatively, the redox activityof the body fluid from a test subject is compared with the redoxactivity of the pooled body fluid samples from non-pregnant femaleswhere the gender specific compatibilities of the ova the femalesgenerated are known, to determine the gender specific compatibility ofthe ovum produced by the test subject. The redox activity of thestandard may be determined using any method encompassed by thisinvention. One standard may be used in the present invention.Alternatively, more than one standard may be used.

The overall redox activity of the body fluid such as urine depends notonly on the composition of the urine, but also, on how the sample isprocessed before assaying, as well as on the assay conditions. Forexample, if the urine sample is not aged or is assayed under mildconditions (see description below), the overall redox activity of theurine correlates with its in vivo overall redox activity. Namely, theurine from a female carrying a female fetus will have a comparativelyhigher overall reducing activity (or antioxidant activity) than urinefrom a female carrying a male fetus.

A similar observation has been made for pre-conception assays. Namely,if the urine sample is not aged or is assayed under mild conditions, theurine sample of a non-pregnant female carrying an ovum compatible withfertilization by an X chromosome-bearing sperm will have a comparativelyhigher reducing activity (or antioxidant activity) than urine from anon-pregnant female carrying an ovum compatible with fertilization by aY chromosome-bearing sperm.

In contrast, if the urine sample is allowed to age and/or is assayedunder harsh conditions (see description below), the urine would exhibitan overall redox activity different from its activity in vivo. Namely,the urine from a female carrying a male fetus will have a comparativelyhigher overall reducing activity than urine from a female carrying afemale fetus.

Likewise, with pre-conception testing, if the urine sample is assayedunder harsh conditions, the urine sample of a non-pregnant femalecarrying an ovum compatible with fertilization by a Y chromosome-bearingsperm will have a comparatively higher reducing activity than urine froma non-pregnant female carrying an ovum compatible with fertilization byan X chromosome-bearing sperm. See Table 1 for the redox activities ofthe urine samples under different processing and assay conditions.

TABLE 1 Urine not aged or assayed Urine aged or Testosterone:Estrogensunder mild assayed under Ratio conditions harsh conditionsPost-conception Male Fetus Greater than Comparatively Comparativelyabout 1 higher overall higher overall oxidizing reducing (or activityantioxidant) activity Female Fetus Less than about 1 ComparativelyComparatively higher overall higher overall reducing (or oxidizingantioxidant) activity activity Pre-conception Ovum Greater thanComparatively Comparatively compatible with about 1 higher overallhigher overall Y chromosome- oxidizing reducing (or bearing spermactivity antioxidant) activity Ovum Less than about 1 ComparativelyComparatively compatible with higher overall higher overall Xchromosome- reducing (or oxidizing bearing sperm antioxidant) activityactivity

The term “mild condition” refers to processing of the body fluid priorto assaying and assaying of the body fluid by treatment at temperatureslower than about 21° C. or treatment with mild chemicals, such as talc,alumina, extraction using organic solvents, solid phase extraction (SPE)chromatography or phosphotungstic acid (PTA). The term “harsh condition”refers to processing of the body fluid prior to assaying or assaying ofthe body fluid by treatment at temperatures greater than about 21° C. ortreatment by harsh chemicals, such as strong acids, strong alkalis,trichloroacetic acid and strong oxidizing agents. Strong oxidizingagents include potassium ferricyanide in the ferric reducing/antioxidantpower (FRAP) assay. The particular condition of the processing of thebody fluid prior to assaying or assaying of the body fluid can bedetermined by one of ordinary skill in the art without undueexperimentation.

There are a number of reducing or oxidizing compounds present in theurine that may interfere with the assays of the present invention.Therefore, the urine may be processed to remove these compounds beforeassaying the redox activity. Interfering reducing and oxidizing agentsinclude urea, creatine, uric acid, ascorbic acid, glucose, glucuronicacid, bilirubin, creatinine, porphyrins and related pigments, nitrites,sulfites, bisulfites, hyposulfites, pyrosulfites, sulfates oxyacids(such as sulfurous acid, bisulfurous acid, hyposulfurous acid,pyrosulfurous acid), urobilinogen, hemoglobin or other interferingproteins, and leucocytes. Interfering agents can be removed by treatmentwith enzymes including ascorbic acid oxidase, glucose oxidase, ureaseand uricase. Glucose may be removed by an anion exchange resin such asAmberlite-4B or alumina. Interfering agents may also includeprogesterone, its derivatives and/or metabolites, which may be removedby adsorbants such as mica and highly oriented pyrolytic graphite, or byantibodies against progesterone, its derivatives and/or metabolites.Ascorbic acid can be removed by resin extraction, diazotized4-nitroaniline-2,5-dimethoxyaniline, ascorbic acid oxidase, leadacetate, iodate, and/or their combinations. Michaelson. Scand J Clin LabInves 20: 97 (1967). Hughes. Analyst. 89:618 (1964).

Chemical, biochemical or physical treatments may also be used to removethe interfering reducing and/or oxidizing compounds before assaying. Thetreatments may include extraction, purification, adsorption, andtreatments by mineral acids, enzymes or microwaving. The body fluid mayalso be processed by treatment with adsorbants and/or precipitants.Adsorbants may be selected from the group consisting of talc,silica-based particles such as silica gel, alumina, florisil, charcoal,kaolin, concanavaline A and its conjugates, calcium phosphate, calciumhydroxide, calcium chloride, Cetyltrimethyl ammonium bromide, lectinspecific to the carbohydrate portion of urinary gonadotropins orglycoproteins, protein or glycoprotein hydrolyzing enzymes, glassfiberfilter, ion exchange resins, affinity ligands, extraction using organicsolvents, solid phase extractants, size exclusion sieves, and reversephase chromatographic materials. Precipitants may be salts of heavymetals, which are selected from the group consisting of barium, lead,molybdenum and tungsten. Precipitants may comprise barium chloride,barium hydroxide mixed with zinc chloride, mercuric chloride, leadacetate and their mixtures. Precipitants may also comprise ammoniumsulphate, dextran, acetonitrile, chloroform, sodium hydroxide,trichloroacetic acid, potassium iodate and their mixtures. One or morethan one adsorbant or precipitant may be used; in addition, adsorbantand precipitant may be used in combination. Adsorbants or precipitantsmay be used to adsorb or retain the interfering reducing or oxidizingagents. Alternatively, adsorbants or precipitants may be used to adsorbor retain desired urinary fraction, which may later be eluted withappropriate solvents for assaying.

When the urine sample is allowed to age, most of the interferingreducing or oxidizing components appear to be eliminated. In oneembodiment, the urine sample is first processed by physical, chemical orbiochemical means to accelerate the aging process. The urine sample isthen treated by other chemical, physical or biochemical means to obtaina purified or partially purified fraction, followed by assaying theoverall redox activity of the urine sample under harsh conditions.

Prior to assaying, insoluble components may be removed from the bodyfluid, for example, by filtration or centrifugation. A non-interferingpH indicator may also be added prior to assaying. The pH of the bodyfluid may be adjusted.

The primary hormones directly linked to fetal gender are sex hormones,namely, testosterone and estrogens. The ratio of testosterone toestrogens is greater than about 1.0 in the urine from a mother carryinga male fetus, whereas the ratio of testosterone to estrogens is lessthan about 1.0 in the urine from a mother carrying a female fetus. Sexhormones, including testosterone and estrogen, are excreted in urine astheir glucuronides associated with large glycoproteins such as humanchorionic gonadotropin (hCG), sex hormone-binding globulin, as well asother glycoproteins. In freshly voided urine, sex hormones maintaintheir association with these large molecules. Without being limited toany specific physiological mechanism, it is believed that the overallredox activity of a urine sample assayed under mild conditions orwithout aging is the net redox activity of various urinary complexeswhich comprise sex hormones and large molecules such as hCG, sexhormone-binding globulin, as well as other glycoproteins.

When urine is assayed under harsh conditions, the hormones which cancontribute to the reducing activity of a urine sample are progesterone,testosterone and human chorionic gonadotropin (hCG) and or itsdegradation byproducts, whereas estrogens can contribute to theoxidizing activity of the urine. hCG is a glycoprotein hormonespecifically produced in pregnancy that is secreted by the embryo soonafter conception and later by the placenta. hCG's major role is tomaintain progesterone production that is critical for maintaining apregnancy in humans. Many currently available early pregnancy tests arebased on the detection or measurement of hCG in the blood or urine. Theurine or blood level of hCG itself does not correlate with the gender ofthe fetus. hCG exhibits reducing activity. Nepomnaschy et al. HumanReproduction. 2008. 23(2): 271. Under harsh assay conditions, if a urinesample is from a female carrying a male fetus, there is no otherstronger reducing agent (or antioxidant) in the urine sample that isable to protect hCG from being oxidized by urinary oxidizing agents.Bowman D. E. J. Biol. Chem. 1940, p 293-302. Gurin et al. J. Biol. Chem.1940. 128: 525. As a result, hCG is oxidized and subsequently degradedto its constituents including galactoses and hexoamines, both of whichhave much stronger reducing activities than hCG. As a result, galactosesand hexoamines contribute to a comparatively higher overall reducingactivity of the urine from a female carrying a male fetus in comparisonwith urine from a female carrying a female fetus. In contrast, in afreshly voided urine sample without aging or under mild assayconditions, if a urine sample is from a female carrying a female fetus,a relatively higher concentration of estrogen in the urine sample isable to protect hCG from oxidizing agents by interacting with theoxidizing agents. As a result, hCG is not oxidized or degraded to thesame extent. Rather, hCG is degraded at a much slower rate in comparisonto hCG in the urine sample from a female carrying a male fetus.Therefore, after aging or under harsh conditions, the urine from afemale carrying a male fetus would exhibit a higher overall reducingactivity than the urine from a female carrying a female fetus.

Sex hormones are excreted in urine as their glucuronide conjugates. Asthe urine sample is aged, glucuronide conjugates are hydrolyzed and freesex hormones released. To facilitate release of the free sex hormonesfrom their glucuronide conjugates, a urine sample may be treated with.the enzyme β-glucuronidases. β-glucuronidases purified from varioussources can be used, including bovine liver, snail Helix pomatia and E.coli. In one embodiment, the urine sample is treated withβ-glucuronidases from E. coli for 24 hours. In another embodiment, theurine sample is treated with E. coli β-glucuronidases for 2 hours.

The urine sample may also be treated by a mineral acid to facilitaterelease of the free sex hormones from their glucuronide conjugates. Themineral acid may be hydrochloric acid or sulfuric acid. The yield ofhydrolysis is determined by the property and concentration of the acid.In one embodiment, a complete hydrolysis of the sex hormone glucuronideconjugates is achieved by treating a urine sample with 3 M sulfuric acidat 37° C. for 24 hours.

The redox potential of the body fluid may also be assayed. Redoxpotential may be determined by measuring the potential differencebetween an inert indicator electrode in contact with the body fluid anda stable reference electrode connected to the body fluid by a saltbridge. The inert indicator electrode acts as a platform for electrontransfer to or from the reference half cell. The indicator electrode maycomprise platinum. The indicator electrode may comprise gold andgraphite. The reference electrode may be stable hydrogen electrode(SHE), Ag/AgCl reference electrode or saturated calomel (SCE) referenceelectrode.

Any suitable assay method, or a combination of two or more such assaymethods, capable of measuring the overall redox activity of the bodyfluid may be used with the methods of the present invention. The overallredox activity of the body fluid may be assayed by reacting the bodyfluid with at least one redox indicator. As used herein, the term “redoxindicator” refers to a molecule that undergoes a measurable change uponbeing reduced or oxidized. A redox indicator may be a reducing agent oroxidizing agent. The measurable change may be changes in color,fluorescence, chemiluminescence, electromagnetic radiation or any othersuitable changes that may be assayed. The amount of the redox indicatorreduced or oxidized may be directly correlated with the overall redoxactivity of the body fluid, which in turn is correlated with fetalgender, or the type of the ovum being produced. The redox indicator maybe a chromogenic chemical capable of changing color upon being reducedor oxidized. The amount of the chromogenic chemical being reduced oroxidized can be measured by the difference in absorbances of the bodyfluid at a specific wavelength before and after the reduction/oxidationreaction.

Below are presented various types of redox assays that may be used todetermine the overall redox activity of the body fluid such as urine.The overall redox activity of the body fluid may be determined bycomparison with a standard where the sex of the fetus is known, or wherethe gender specific compatibility of the ovum is known. The comparativelevels of the redox activity of the urine samples are shown in Table 1.The particular condition in each assay may be determined by a person ofordinary skill in the art without undue experimentation.

The overall reducing activity of a body fluid may be assayed using aheteropoly acid or its corresponding salts. Heteropoly acids are a classof acids each comprising a particular combination of metal, hydrogen,oxygen and other non-metal atoms. Specifically, a heteropoly acidcontains a metal termed addenda atom, such as tungsten, molybdenum orvanadium; oxygen; a element termed hetero atom generally from thep-block of the periodic table, such as silicon, phosphorus or arsenic;and acidic hydrogen atoms. Seelhttp//en.wikipedia.org/wiki/Heteropoly_acid [Online. Retrieved on Mar.9, 2009]. Two of the better known heteropoly acids are phosphotungsticacid (PTA) with the formula H₃PW₁₂O₄₀ and phosphomolybdic acid with theformula H₃PMo₁₂O₄₀.

The phosphotungstic and phosphomolybdic acids are very sensitive toreduction, and yield highly colored compounds even upon moderatereduction. Wu. Contributions to the chemistry of phosphomolybdic acid,phosphotungstic acid and allied substances. J.

Biol. Chem. 1920. XLIII, 1: 189-220. Under acidic condition, PTAassociates with proteins and forms precipitates in solution.Phosphotungstate: a “universal” (nonspecific) precipitant for polarpolymers in acid solution. Journal of Histochemistrv and Cvtochemistry.J. E. Scott. 1971. 19, 11, 689. Besides proteins, PTA also has anaffinity for carbohydrates at low pH. Pease DC. J. Ultrastucture Res.1966. 15: 555. As pH of its solution increases, a heteropoly acidgenerally decomposes to its simpler constituent acids, but can beregenerated by re-acidification.

The assay using PTA may be conducted under mild conditions. With a PTAassay, the urine from a female carrying a female fetus will have acomparatively higher overall reducing activity as compared with urinefrom a female carrying a male fetus. When a urine sample from a pregnantfemale carrying a male fetus contacts PTA, PTA remains intact in theurine and forms insoluble complexes with urinary proteins andglycoproteins. Thus, this urine sample appears as a white colloidalsuspension in the PTA assay for at least about 12 hours.

In contrast, for urine from a pregnant female carrying a female fetus,although PTA initially forms insoluble complexes with urinary proteinsand glycoproteins, the complexes gradually decrease as PTA is beingreduced by the urine. The reaction mixture becomes transparent overtime. The time period for the reaction mixture to turn from a whitecolloidal suspension to clear solution may vary among different urinesamples. In some embodiments, the time period may range from about 5minutes to about 12 hours. Shorter or longer time periods may also beappropriate, such as about 1 minute, 2 minutes, 3 minutes, 4 minutes, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours and 20hours.

Tungsten or molybdenum is preferred as the addenda atom of theheteropoly acid or its salts used in the present invention. A heteropolyacid or its salts may consist of one or more than one types of addendaatoms. The combinations of the addenda atoms include molybdenum andtungsten, vanadium and molybdenum, and vanadium, molybdenum andtungsten. The hetero atom of a heteropoly acid or its salts is selectedfrom the group consisting of phosphorus, arsenic, silicon and germanium.Phosphorus is preferred as the hetero atom. Heteropoly acids or theirsalts comprising molybdenum and phosphorus, molybdenum and arsenic, ortungsten and phosphorus are preferred embodiments. A heteropoly acid orits salts may be selected from the group consisting ofphospho-12-molybdic acid, phospho-18-molybdic acid, 12-molybdoarsenicacid, 18-molybdoarsenic acid, 11-molybdo-1-vanadophosphoric acid,10-molybdo-2-vanadophosphoric acid, phospho-12-tungstic acid,phospho-18-tungstic acid, phospho-24-tungstic acid and9-molybdo-3-vanadophosphoric acid. Phospho-18-molybdic acid,phospho-12-tungstic acid, phospho-18-tungstic acid andphospho-24-tungstic acid are preferred embodiments. Heteropoly acids andtheir salts may be used individually or their combinations may be used.

The overall redox activity of a body fluid may be assayed by achromogenic chemical comprising an oxidation-reduction sensitivemetallic ion. The metallic ion may be selected from the group consistingof copper, iron, chromium, tungsten, molybdenum ions in an oxidizedstate.

The chromogenic chemical may be a metal-organic complex which, due toits extremely high oxidizing activity (correlated with high redoxpotential), can be reduced by many reducing agents and undergo change incolor. The ferric reducing/antioxidant power (FRAP) assay can be used toassay the overall reducing activity of a body fluid. The ferrictripyridyltriazine (Fe(III)-TPTZ) complex is reduced under acidicconditions by a reducing agent to form ferrous tripyridyltriazine(Fe(II)-TPTZ). Fe(III)-TPTZ is yellow in color. The production ofFe(II)-TPTZ is easily detectable and measurable because of its intenseblue color with a maximum absorption at wavelength 593 nm.

The FRAP assay may be conducted under harsh conditions, where the urinefrom a female carrying a male fetus will have a comparatively higheroverall reducing activity than urine from a female carrying a femalefetus. Accordingly, a urine sample from a female carrying a male fetusis able to reduce Fe(III) to Fe(II), or Fe(III)-TPTZ to Fe(II)-TPTZ,with the reaction mixture turning dark blue. In contrast, a urine samplefrom a female carrying a female fetus is not able to reduce Fe(III), orFe(III)-TPTZ, and thus, the reaction mixture remains yellow.

Potassium ferricyanide may also be used in the FRAP assay. Oyaizu.Studies on products of Browning reaction. J. Nutrition. 44: 307-315(1986). Under the harsh conditions of the FRAP assay, the urine from afemale carrying a male fetus will have comparatively higher overallreducing activity than urine from a female carrying a female fetus.Accordingly, a urine sample from a female carrying a male fetus is ableto reduce potassium ferricyanide to potassium ferrocyanide, which isthen able to form dark blue product when ferric chloride is added. Incontrast, a urine sample from a female carrying a female fetus is notable to reduce potassium ferricyanide, and, thus, the reaction mixtureremains golden yellow or faint green in color.

For fraternal multiples with different genders, when the FRAP assay isconducted, the urine with a comparatively higher overall reducingactivity than urine from a female carrying a female fetus correlateswith at least one male fetus, whereas the urine with comparable overallreducing activity to urine from a female carrying a female fetuscorrelates with at least two female fetuses.

The overall reducing activity of a body fluid may be assayed by thereduction of Cu²⁺ to Cu⁺, which may be detected by the complex formationbetween Cu⁺ and bathocuproine. The amount of Cu⁺ being generatedcorrelates with the overall reducing activity of the body fluid. TheCu⁺-bathocuproine complex is stable and has a maximum absorption atwavelength around 480-490 nm. In one embodiment, the amount of Cu⁺ beinggenerated is quantified by referring to a standard curve, which usesuric acid as the reducing agent.

The overall reducing or antioxidant activity of the body fluid may beassayed by its ability to capture a free radical in the presence of aredox indicator. In one embodiment, the radical is hydroxy radical (OH.)generated either by a hydroxyl radical-generating system or internallywithin the body fluid, with sodium salicylate as the redox indicator. Inthe absence of other antioxidant (or reducing agent), the colorlesssalicylate ion reacts with OH. to produce dihydroxybenzoic acid, whichis blue in color and has a maximum absorption at wavelength 510 nm. Whenother antioxidant (or reducing agent) is present, hydroxyl radical issequestered or trapped by this antioxidant (or reducing agent) instead.

As a result, sodium salicylate stays intact and the reaction mixtureremains colorless.

In another embodiment, the overall redox activity of the body fluid maybe assayed by its ability to facilitate the generation of free radicals.The assay reagents may comprise sodium salicylate and a hydroxyl radicalgenerating system including a ferric compound, such as ferric sulfate,and H₂O₂. When the reducing agent is present, it reduces ferric toferrous, which results in further reduction of H₂O₂ to generate hydroxylradical OH* through Fenton reaction. Salicylate ion then reacts with OH.to produce dihydroxybenzoic acid that is blue in color. The reaction isdiagramed below.

Fe³⁺+Reducing Agent→Fe²⁺+Oxidized Reducing Agent

Fe²⁺+H₂O₂→Fe³⁺+OH.+OH—

Sodium Salicylate+20H.→Dihydroxybenzoic Acid

In the absence of the reducing agent, no OH. is generated. As aconsequence, sodium salicylate remains colorless.

The overall redox activity of the body fluid may also be detected usingredox sensitive polymers such as polyaniline. Polyaniline is polymerizedfrom the aniline monomer and has the following structure,

where x is half the degree of polymerization. See,http://en.wikipedia.org/wiki/Polyaniline [Online. Retrieved on Mar. 6,2009]. Polyaniline can be found in one of three oxidation states. Theleucoemeraldine form of polyaniline (n=1, m=0) is the fully reducedstate and is white or colorless. Pemigraniline (n=0, m=1) is the fullyoxidized state and is blue or violet. The emeraldine (n=m=0.5) form ofpolyaniline is the intermediate state and is green or blue. The colorchange associated with polyaniline in different oxidation states may beexploited to use polyaniline as a redox indicator. Huanga et al.Development and characterization of flexible electrochromic devicesbased on polyaniline and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid). Electrochimica Acta. 51, 26: 5858-5863 (2006).

In another embodiment, the overall reducing activity of a body fluid maybe assayed by an in situ methodology. For example, a redox indicator,which may be selected from the group consisting of thionine, toluidineblue O and cresyl violet, is immobilized to agarose beads. The agarosebeads are then packed into a flow cell. The body fluid is allowed topass through the flow cell and the redox states of the redox indicatoris monitored by spectrophotometry. Jones. Evaluation of immobilizedredox indicators as reversible, in situ redox sensors for determiningFe(III)-reducing conditions in environmental samples. Talanta. 2001. 55,4: 699-714.

The overall redox activity of a body fluid may be detected by generalreducing sugar tests using copper ion, neocuproine or alkalineferricyanide method with or without color formation witho-phenanthroline complex. Prado et al. Phytochem. Analysis. 9 (2): 58-62(1998).

The overall redox activity of the body fluid may also be detected by theOxygen Radical Absorbance Capacity (ORAC) assay, which measures bothlipophilic and hydrophilic antioxidant capacity using the same peroxylradical generator. The ORAC assay measures the oxidative degradation ofa fluorescent molecule (such as beta-phycoerythrin or fluorescin) afterbeing mixed with free radical generators such as azo-initiatorcompounds. Azo-initiator compounds produce peroxyl free radical, whichdamages the fluorescent molecule resulting in loss of fluorescence.Antioxidant or reducing agent such as estrogen is able to protect thefluorescent molecule from the oxidative degeneration. The degree ofprotection by the antioxidant or reducing agent may be quantified usinga fluorometer. Williams et al. A comparison of mammalian and plantestrogens on vascular reactivity in young and old mice with or withoutdisruption of estrogen receptors. Current Topics in Nutraceutical Res.2, 4: 191 (2004). See also,http://en.wikipedia.org/wiki/Oxygen_Radical_Aborbance_Capacity [Online.Retrieved on Mar. 2, 2009].

The overall redox activity of the body fluid may be detected by theAntioxidant Capacity (AOC) assay using the3-ethylbenzothiazoline-6-sulfonate (ABTS) system or TPTZ. Bahramikia etal. A comparison of Antioxidant Capacities of ethanol extracts of S.Hortensis and A. Dracunculus leaves. Pharmacology Online 2: 694-704(2008).

The overall redox activity of the body fluid may be detected by the useof immobilized chromogenic radicals. In one embodiment, either of twostable lipophylic chromogenic radicals, DPPH(2,2-diphenyl-1-picrylhydrazyl) radical and GV (2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexanedien-1-ylidene)-p-tolyloxy)radical, is immobilized in plasticized polymer films, such as polyvinylchloride (PVC) films. When the polymer film containing immobilized2,2-diphenyl-1-picrylhydrazyl (DPPH) radical is reduced by a body fluid,it changes color irreversibly from purple (maximum absorption atwavelength 520 nm) to yellow. Steinberg et al. Chromogenic radical basedoptical sensor membrane for screening of antioxidant activity. Talanta8: 15 (2006).

The overall redox activity of the body fluid may be detected byimmunoassay such as immunofluorescence, using antibodies against genderrelated molecules; chemiluminescence; bioluminescence such as greenfluorescent proteins; antibody-tagged gold nanoparticles of differingsizes; chemical tagging or fluorescent dyes. Kohen et al. Recentadvances in chemiluminescence-based immunoassays for steroid hormones.J. Steroid Biochem. 27, 1-3: 71-79 (1987).

The overall oxidizing activity of the body fluid may be assayed byadding a source of ferrous ions to the body fluid, whereby oxidants inthe sample oxidize at least a portion of the ferrous ions to ferricions. Then a chromogenic compound is added to the sample, which reactswith at least a portion of the ferric ions. U.S. Patent Publication No.20040259186.

The overall reducing activity of the body fluid may be assayed usingreagent selected from the group consisting of ferricyanide salts,dichromate salts, permanganate salts, vanadium oxides,dichlorophenolindophenol, osmium bipyridine complexes, and quinones. Theoverall oxidizing activity of the body fluid may be assayed usingreagent selected from the group consisting of iodine, triiodide salts,ferrocyanide salts, ferrocene, [Cu(NH3)₄]²⁺ salts and [Co(NH3)₆]³⁺salts. The redox indicator of the present methods may be selected fromthe group consisting of 2,6-dichlorophenolindophenol (DCPIP),3,3′,5,5′tetramethylebenzidine (TMB), 1,4-phenylenediamine (DMPDA),Phenanthridine, 2,6-Dichloroindophenol,2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid,N,N-Dimethylphenylenediamine, 2-Amino-p-cresol (APC), Xylenol orange,8-Hydroxy-7-iodo-5-quinoline-sulfonic acid and4,5-Dihydroxy-1,3-benzene-di-sulfonic acid.

The present invention also provides a method for pre-conception babygender planning by assaying the overall redox activity of a female'surine or other body fluid. The body fluid may be obtained anytime duringa menstrual cycle, but preferably around the time of ovulation. Withoutbeing limited to any specific physiological mechanism, it is believedthat there are two types of ova, each of which, in most of ovulatingfemales, is generated in alternate months. One type of ovum iscompatible with fertilization by a Y chromosome-bearing sperm to producea male fetus, while the other type of ovum is compatible withfertilization by an X chromosome-bearing sperm to produce a femalefetus. The overall redox activity of the maternal urine correlates withthe gender specific compatibility of the ovum released in a particularmenstrual cycle.

Under harsh assay conditions or with aging, the urine sample of anon-pregnant female carrying an ovum compatible with fertilization by aY chromosome-bearing sperm will have comparatively higher reducingactivity than urine from a non-pregnant female carrying an ovumcompatible with fertilization by an X chromosome-bearing sperm (seeTable 1 for summary). It is believed that a female produces two types ofova which generally alternate every month, i.e., if in one month theovum being produced is compatible with fertilization by an Xchromosome-bearing sperm, then in the next month the ovum being producedis compatible with fertilization by a Y chromosome-bearing sperm. Thepre-conception urine assay can help a couple conceive a baby having adesired gender. For example, if a couple desires a baby girl, it isrecommended that they plan to conceive in any month when the ovum beingproduced is compatible with fertilization by an X chromosome-bearingsperm. Similarly, if a couple wishes a baby boy, it is recommended thatthey plan to conceive in any month when the ovum being produced iscompatible with fertilization by a Y chromosome-bearing sperm. Thepurpose of the present pre-conception methods is to raise the chances ofconceiving the baby of desired gender by advising appropriate menstrualcycles for conception.

The present invention provides a method for pre-conception baby genderplanning comprising the steps of: (a) contacting a body fluid from anon-pregnant female with at least one redox indicator; (b) measuringredox activity of the body fluid; and, (c) comparing the redox activityof the body fluid with at least one standard to determinegender-specific compatibility of the ovum released in a menstrual cycle.The body fluid may be obtained near ovulation or middle of the menstrualcycle. The body fluid may be processed prior to prior to step (a).

If two urine samples collected in two consecutive months manifestdifferent redox activities, it suggests that the female has a regularalternating ovulation pattern as described above. If both urine samplesdemonstrate comparatively higher reducing activity than urine from anon-pregnant female carrying an ovum compatible with fertilization by anX chromosome-bearing sperm, the female may have an irregular ovulationpattern which is difficult to normalize according to the presentinvention. If both urine samples demonstrate comparatively higheroxidizing activity than urine from a non-pregnant female carrying anovum compatible with fertilization by a Y chromosome-bearing sperm, thefemale's irregular ovulation pattern may be normalized through diet andexercise.

As described above, any suitable assay method, or a combination of twoor more such assay methods, capable of measuring the overall redoxactivity of the body fluid may be used with the present pre-conceptionassay or the present post-conception assay.

The overall redox activity of the body fluid during a menstrual cyclewithin which a female conceives is similar to the redox activity of herbody fluid after conception. Namely, if her pre-conception body fluidhas a comparatively higher overall reducing activity than that of thebody fluid from a non-pregnant female carrying an ovum compatible withfertilization by an X chromosome-bearing sperm under the harsh condition(for example, in the FRAP assay), her ovum is compatible withfertilization by a Y chromosome-bearing sperm. Consistently, herpost-conception urine sample would also have a higher overall reducingactivity than that of urine from a female carrying a female fetus, whichis correlated with a male fetus. Conversely, if her pre-conception urinesample has a higher overall oxidizing activity than that of urine from anon-pregnant female carrying an ovum compatible with fertilization by aY chromosome-bearing sperm under harsh conditions, her ovum iscompatible with fertilization by an X chromosome-bearing sperm.Likewise, her post-conception urine sample would also have a higheroverall oxidizing activity than that of urine from a female carrying amale fetus, which is correlated with a female fetus.

The present invention further provides a method for determining thegender of a fetus that is within a female's uterus using remotedetection devices. In vivo, a male fetus as a tissue mass has a higheroverall oxidizing activity, or a lower reducing activity, than thesurrounding maternal tissues, while a female fetus has a higher overallreducing or antioxidant activity than the surrounding maternal tissues.As is the case with post-conception or pre-conception urine tests, theredox activity of the fetal tissue or maternal tissue may be comparedwith a standard where the fetal gender is known. The remote detectiondevice of the present invention may comprise a probe. The device maydetect the redox activity of different parts of a mother's internaltissues by non-invasively sensing the internal electrochemical signals,electromagnetic signals, or any other suitable physical and/or chemicalsignals. In one embodiment, internal redox potentials are detected bythe remote detection device. In another embodiment, internal electroniccharges are detected by the remote detection device. In a furtherembodiment, the remote detection device comprises an electroscope todetect electrochemical potential of fetal and maternal tissues.

The present remote detection device may comprise broadband diffuseoptical spectroscopy. Lee et al. Noninvasive in vivo monitoring ofmethemoglobin formation and reduction with broadband diffuse opticalspectroscopy. J. Appl. Physiology 100: 615-622. As known in the art,oxidation of proteins and amino acids generates ultra weak photonemission. The present remote detection device may comprise a sensitivephotomultiplier system to detect the ultra weak photon emission that isassociated with a male or female fetus (Noninvasive monitoring ofoxidative skin stress by ultra weak photon emission (UPE) measurement[online], [retrieved on Mar. 2, 2009]. Retrieved from the Internet <URL:http://www.find-health-articles.com/rec_pub_(—)18211608-non-invasive-monitoring-oxidative-skin-stress-ultraweak-photon.htm>).The present remote detection device may comprise electron paramagneticresonance imaging (EPRI) by using redox sensitive paramagnetic contrastagents. Yamada et el. Feasibility and assessment of non-invasive in vivoredox status using electron paramagnetic resonance imaging (EPRI). ActaRadiol. 43 (4):433-40 (2002). The present remote detection device maycomprise measuring the surface oxygen tension of organs. Miller A. T.Jr. University of North Carolina at Chapel Hill. Tissue oxygen tensionand the intracellular oxidation-reduction state. Personal communication.Other suitable means may also be used for the present remote detectiondevice. See, Wang. Remote electrochemical sensors for monitoring organicand inorganic pollutants. Trends in Anal. Chem. 16: 84 (1997). Balcom etal. Spatial and temporal visualization oftwo aqueous ironoxidation-reduction reactions by nuclear magnetic resonance imaging. J.Chem Soc. Chem. Comm. 1992: 312-313. Livesey et al. Measurement oftissue oxidation-reduction state with carbon-13 nuclear magneticresonance spectroscopy. Cancer Research. 49: 1937-1940 (1989).

The change of the redox indicator may be analyzed with a scientificinstrument, such as spectrophotometer, fluorometer, turbiditimeter,luminometer, fluorescence meter or colorimeter. The change in color ofthe chromogenic molecule may also be observed visually. The change ofthe redox indicator may be converted to digital signals which can beprocessed by digital, electronic or other sensing scientific instrumentsor materials. The scientific instrument may convert the assay results toa digital text response using any suitable means such as an electronicprogramming. In one embodiment, the instrument shows the text “It's aBOY” when the assay results suggest that the body fluid is from a femalecarrying a male fetus, and “It's a GIRL” when the assay results suggestthat the body fluid is from a female carrying a female fetus. Similarly,for a pre-conception assay on the body fluid from a non-pregnant female,the instrument may show “Time for a baby boy” if the ovum produced in aparticular menstrual cycle is compatible for fertilization by a Ychromosome-bearing sperm, and “Time for a baby girl” if the ovumproduced in a particular menstrual cycle is compatible for fertilizationby an X chromosome-bearing sperm.

The overall redox activity of the body fluid may be assayed using anelectrochemical sensor, which is able to produce an electrochemicallydetectable signal upon being reduced or oxidized. In this case, adetection device may be used which comprises the redox indictor and hasa disposable electrochemical cell, such as a thin layer electrochemicalcell. The sensing electrode may consist of platinum, palladium, carbon,indium oxide, gold, iridium, copper, steel or mixture thereof. U.S. Pat.No. 6,638,415. WO2002/006806. Carbon nanotubes may be used forelectrochemical sensing. Carbon nanotubes have the ability to promoteelectron transfer reactions when used as an electrode material inelectrochemical reactions. U.S. Patent Publication No. 20060096870.Electrochemistry and electrogenerated chemiluminescence with a singlefaradaic electrode may be used for the present methods. It comprises ofa faradaic working electrode and a capacitive counter electrode. Whenelectrical energy is supplied in the presence of a body fluid, afaradaic charge transfer occurs wherein at least one of (i) light, (ii)current, (iii) voltage and (iv) charge can be measured to determine thepresence or amount of analyte in a sample. WO2007/053191.

The overall redox activity of the body fluid may be assayed using anysuitable chemical, physical, biochemical or biological means. Theoverall redox activity of the body fluid may be assayed by contactingthe body fluid with at least one microorganism such as a bacterium, andobserving the physiological responses such as generation of coloredproduct, movement of the bacterium's cilia. The overall redox activityof the body fluid may be assayed by redox-sensitive green fluorescentprotein (GFP). Dooley et al. Imaging dynamic redox changes in mammaliancells with GFP indicators. J. Biol. Chem. 279, 24: 22284-22293 (2004).The overall redox activity of the body fluid may be measured by assayingfluorescence. In one embodiment, 5-cyano 2-3 ditolyl tetrazoliumchloride (CTC) is used for the present assay. CTC's oxidized form, whichis colorless and non-fluorescent, is readily reduced to fluorescent,insoluble CTC-formazan form.

The present methods may be modified to allow many samples to be testedand results read simultaneously using, for example, a microplate ormicroarray.

The present invention also provides an article of manufacture such as akit comprising (a) a urine collecting vial; (b) a solid substratecomprising a redox indicator, wherein the redox indicator comprises atleast one chromogenic chemical; (c) printed material instructing a womanto collect urine and contact the urine with the solid substrate; and (d)printed material instructing a woman to compare redox activity of theurine sample with at least one standard to determine the gender of theunborn child based on change in color in the chromogenic chemical. Thekit may also comprise other chemicals for processing and/or assaying theoverall redox activity of the body fluid. The printed matter may alsoindicate that, to determine the gender of an unborn child, the enclosedchemicals are to be used to assay the overall redox activity of the bodyfluid which will be compared with a standard. The printed material mayinstruct processing the urine prior to contact the urine with the solidsubstrate. The printed matter provides instructions as to how to use theenclosed chemicals for the test. Pictorial depictions of theinstructions may be included in the printed matter. The printed mattermay indicate that the body fluid is to be collected at any time during apregnancy, starting from the first day of missed menstruation. Theprinted matter may also indicate that optimum results may be obtained ifthe body fluid is collected between the 5th and 15th week of pregnancy.The printed matter may include color standards for resultinterpretations and indicates the color (or a range of colors) thatcorrelates with a male (or female) fetus. The chemicals may be providedin pre-measured quantities and may be in the form of solution, solidcrystals or dry film. The chemicals may be placed in capped vials orbottles and placed in separate compartments. The chemicals may beabsorbed or retained to solid supports and arranged sequentially to forma trickling column so as to ensure that various processes are conductedin a desired order. A kit may contain chemicals in sufficient amountsfor one, two or more assays.

The present invention further provides an article of manufacture such asa kit comprising necessary chemicals for processing and/or assaying theoverall redox activity of the body fluid such as urine, as well asprinted matter indicating that, to determine the suitable menstrualcycle for conceiving a baby of desired gender, the enclosed chemicalsare to be used to assay the overall redox activity of the body fluid inat least two consecutive menstrual cycles. The printed matter mayindicate that the body fluid may be collected at any time during amenstrual cycle. The printed matter may also indicate that optimumresults may be obtained if the body fluid is collected around theovulation period. The printed matter provides instructions as to how touse the enclosed chemicals for the test. The printed matter alsoincludes color standards for result interpretations and indicates thecolor (or a range of colors) that correlates with an ovum compatiblewith fertilization by a Y chromosome-bearing sperm or an ovum compatiblewith fertilization by an X chromosome-bearing sperm. The printed matterindicates that two types of ova are produced in alternate months, andsuggests that a woman who wishes to have a baby boy (or girl) try toconceive in alternate months, starting from the month an ovum compatiblewith fertilization by a Y chromosome-bearing (or X chromosome-bearing)sperm is ovulated.

The redox activity may be measured on a solid substrate which comprisesa redox indicator, such as a chromogenic chemical. The chromogenicchemical may be impregnated on a strip. The article of manufacture ofthe present invention may be a test strip. The test strip may have asample application site where the body fluid may be applied. The bodyfluid may then pass through various aging, processing, filtering,adsorbing or chromatographic media, which ultimately leads to theprocedure of assaying the overall reducing or oxidizing activity of thesample by reacting with at least one redox indicator. The test strip mayhave more than one sample application site so that the body fluid may beapplied at more than one site on the strip at the same time. The samebody fluid applied at different sites on the strip may undergo differenttreatments and may meet different chromogenic chemicals or redoxindicators. The overall reducing or oxidizing activity of the sample isthen evaluated based on a specific pattern of colors or other measurablesignals.

The methods of the present methods may be used to test a body fluidobtained from any mammal, such as a human, horse, dog, monkey, sheep,pig and cat.

The Examples illustrate embodiments of the invention and are not to beregarded as limiting.

EXAMPLE 1 Post-Conception Assay

Table 2 tabulates the results for post-conception tests conducted for4,524 women during the period between 1999 and 2008, with an averageobserved accuracy of about 90%.

At the initial stage, i.e., from the years 1999 through mid-2003, urinesamples were tested using sodium salicylate as the redox indicatorcombined with spectrophotometry (see Example 4). PTA test (see Example2) was started in 2002. FRAP assay (see Example 3) has been conductedsince 2008.

TABLE 2 Stage of pregnancy Boys Boys Girls Girls Accuracy for Accuracyfor YEAR (wks) Observed Reported Observed Reported Boys % Girls %1999-2001 7 212 242 229 258 87.6 88.8 8 305 344 331 367 88.7 90.2 9 299338 332 378 88.5 87.8 10 & up 281 328 293 345 85.4 84.9 2002 6 22 29 2325 75.9 92.0 7 21 31 18 19 67.7 94.7 8 32 36 19 21 88.9 90.5 9 17 21 2022 81.0 90.9 10 & up 46 54 26 31 85.2 83.9 2003 6 14 16 26 31 77.8 83.97 26 33 16 18 78.8 88.9 8 25 25 22 24 100.0 91.7 9 16 16 13 13 100.0100.0 10 & up 37 45 20 21 82.2 95.2 2004 6 16 19 13 15 84.2 86.7 7 26 3519 22 74.3 86.4 8 22 29 22 24 75.9 91.7 9 9 12 12 14 75.0 85.7 10 & up16 21 26 31 76.2 83.9 2005 5 10 11 5 5 90.9 100.0 6 13 14 13 14 92.992.9 7 21 26 20 24 80.8 83.3 8 8 9 9 10 88.9 90.0 9 9 10 12 13 90.0 92.310 & up 48 58 48 51 82.8 94.1 2006 5 22 23 20 22 95.7 90.9 6 30 33 24 2590.9 96.0 7 13 13 15 15 100.0 100.0 8 15 15 11 13 100.0 84.6 9 8 8 8 9100.0 88.9 10 & up 26 28 39 41 92.9 95.1 2007 5 19 21 32 36 90.5 88.9 616 18 13 15 88.9 86.7 7 16 18 17 18 88.9 94.4 8 20 24 15 15 83.3 100.0 912 13 12 14 92.3 85.7 10 & up 39 42 45 48 92.9 93.8 2008 5 26 28 34 3792.9 91.9 6 25 25 39 42 100.0 92.9 7 22 23 30 32 95.7 93.8 8 23 25 21 2292.0 95.5 9 28 30 25 25 93.3 100.0 10 & up 47 49 55 58 95.9 94.8 TOTAL1,958 2,241 2,042 2,283 88% 91.3% Total Fetal Gender 4,524 AverageObserved Predicted Accuracy: 90%

From 1999 to 2001 we tested urine samples collected after 21 days pastmissed menstruation date. Since 2005, we have been testing urine samplescollected 1 day after missed menstruation, i.e., the 5th week ofpregnancy.

If the urine samples were aged for a longer period of time, the accuracyof the present methods could approach 100%. However, from a clinicalperspective, aging the urine samples for long period of time is notapplicable. Accordingly, techniques to accelerate the aging process byphysical, chemical or biochemical means were developed.

EXAMPLE 2 Processing of Urine Samples for the PTA Assay

For the PTA assay, the urine samples were collected and processed asfollows.

Post-conception urine samples were collected from about 2,782 females atvarious time points of a pregnancy ranging from 4 days before missedmenstruation to the 20th week of pregnancy. The customers were shipped avial for urine sample collection with a boric acid tablet in the vial asa preservative. Customers were asked to collect urine voided during aresting period including early morning for at least two days. Customerswho were working at night were asked to collect urine during day-timeinstead for three consecutive resting periods. The samples were returnedto the clinic by mail which took from 2 to 5 days depending upon thedistance. The delivery of the urine sample by mail provided a timeperiod for sample aging. The urine sample may be further aged at roomtemperature for longer periods of time.

The urine was then processed as follows. 2 ml of the urine sample waspassed through acidic alumina to eliminate significant amount ofpigments and glucose. A C 18 column was activated by methanol andpre-equilibrated with water. The cleared urine sample was then loaded onthe C18 column to be subjected to solid phase extraction (SPE). Afterthe urine sample passed through the C18 column, about 95% of sexhormones were retained on the column. The fraction of the urine samplethat passed through or did not bind to the column was referred to asFraction A. Fraction A was then treated with 50 ul of 10% Na₂HPO₄ and anexcess amount of calcium phosphate (about 160 mg) to remove bilirubin,urobilinogen, porphyrins, and other pigments. 50 ul of saturatedmercuric chloride was added to Fraction A to precipitate uric acid,ascorbic acid, creatine and some interfering proteins. 50 ul of 10% leadacetate solution was then added to Fraction A to remove creatine, someinterfering proteins and bilirubin. 10 mg of barium chloride crystalswas added to Fraction A to remove bilirubin, remaining interferingproteins and some interfering inorganic oxidants/reductants. The pH ofFraction A was then adjusted to 8.5 with 50 ul to 70 ul of 1 M sodiumhydroxide, followed by centrifugation or filtration to remove theinsoluble components.

In the PTA test, 20 mg of crystalline phosphotungstic acid was added to0.75 ml of Fraction A, and the solution was kept below 21° C. for 12hours. If the solution turned to a stable white fluorescent colloidalsuspension after 12 hours, the urine sample was reported to be from afemale carrying a male fetus. If the solution initially formed whiteprecipitates which later disappeared to result in a transparentsolution, the urine sample was reported to be from a female carrying afemale fetus. Repeated analysis of the same urine sample aged fordifferent periods of time improved the accuracy of the analyticalresults.

EXAMPLE 3 Processing of Urine Samples for a FRAP Assay

For the FRAP assay, the urine samples were processed as follows. Theurine samples were treated as above in Example 2; however, in this casethe components that were retained on the C18 column were eluted andtested.

2 ml of the urine sample was passed through acidic alumina to eliminatesignificant amount of pigments and glucose. A C18 column was activatedby methanol and pre-equilibrated with water. The cleared urine samplewas then loaded on the C18 column. After the urine sample passed throughthe C18 column, about 95% of sex hormones were retained on the column,which were than eluted with 1 ml of HPLC grade methanol and calledFraction B. Fraction B was diluted with 2 ml water. 2.5 ml of dilutedFraction B was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50° C.for 20 min and cooled to room temperature. The mixture was then mixedwith 2.5 ml of 10% trichloroacetic acid and centrifuged at 6500 rpm for10 min. The supernatant (2.5 ml) was mixed with 2.5 ml distilled water,before 0.5 ml of 0. 1 % ferric chloride was added. In this assay, theresults were read within one minute after the addition of the ferricchloride, as the results were not stable at later time points.

If Fraction B turned to deep blue after the FRAP assay, the urine samplewas reported to be from a female carrying a male fetus. If Fraction Bturned to golden yellow, faint yellow or faint green after the FRAPassay, the urine sample was reported to be from a female carrying afemale fetus. Repeated analysis of the same urine sample aged fordifferent periods of time improved the accuracy of the analyticalresults.

If the same urine sample was analyzed by more than one type of assay, wehave found that the accuracy of the results can approach 100%. Forexample, if the PTA test on Fraction A produced a stable whitefluorescent colloidal suspension after 12 hrs, and the FRAP test onFraction B resulted in a deep blue solution, it was almost 100% accuratethat the urine sample was associated with a male fetus. Similarly, ifthe PTA test on Fraction A produced a transparent solution after 12 hrs,and the FRAP test on Fraction B resulted in a solution with goldenyellow, faint yellow or faint green in color, the accuracy was close to100% that the urine sample was correlated with a female fetus.

Alternatively, the FRAP test can also be performed on Fraction A. Whenurine samples were aged for at least 3 months at room temperature, theaccuracy of this method conducted was close to 100%.

EXAMPLE 4 Redox Activity Assay with Sodium Salicylate

The urine samples were processed and assayed using sodium salicylate asfollows.

For post-conception urine sample assays, customers were asked to collectearly morning urine samples on three consecutive mornings and drop themoff at the clinic. All three samples from the same customer were studiedseparately and a report was issued after pooling the results. To processthe urine sample, 4 ml of urine sample was treated with 20 mg of leadacetate for 30 minutes to precipitate interfering proteins. 1 ml ofprocessed urine sample and 1 ml of 0.75 M hydrochloric acid were addedto a 10 ml glass vial, before 1 ml of 1% sodium salicylate was addedslowly to the vial which was gently shaken to mix the reagents. Controlsfor the test were also included where, instead of 1 ml of 1% sodiumsalicylate, Iml of distilled water was added to the vial. The reactionmixture was incubated at 50° C. for 2 hours and then cooled to roomtemperature. The absorbance of the reaction mixture at wavelengthsbetween 470 nm to 550 nm was then measured against control in aspectrophotometer. When a peak was observed around wavelength 510 nm, aresult of a male fetus was recorded for this sample. When a trough or astraight base line was observed around wavelength 510 nm, a result of afemale fetus was recorded for this sample.

EXAMPLE 5 FRAP Assay with Fe(III)-TPTZ

Fe(III)-TPTZ may also be used as the redox indicator for the FRAP assay.Under acidic conditions, Fe(III)-TPTZ complex can be reduced toFe(II)-TPTZ. Fe(II)-TPTZ has intense blue color and can be monitored atwavelength 593 nm. FRAP working solution was freshly prepared by mixing10 ml 300 mmol/1 acetate buffer (pH 3.6), 1.0 ml 10 mmol/1 TPTZ (2,4,6tripyridyl-s-triazine) in 40 mmol/1 HCl solution, and 1.0 ml 20 mmol/1FeCl₃.6H₂O solution.

2 drops of processed urine sample were added to 0.5 ml FRAP workingsolution and the solution was incubated for 10 minutes. Under the harshconditions of the FRAP assay, the urine from a female carrying a malefetus has a comparatively higher overall reducing activity than urinefrom a female carrying a female fetus. Accordingly, a urine sample froma female carrying a male fetus was able to reduce Fe(III)-TPTZ toFe(II)-TPTZ, with the reaction mixture turned dark blue. In contrast, aurine sample from a female carrying a female fetus was not able toreduce Fe(III)-TPTZ, and thus, the reaction mixture remained yellow.When urine samples were aged for at least 3 months at room temperature,the accuracy of this method conducted on 161 urine samples was close to100%. Among the 161 urine samples, 67 samples were from females carryinga male fetus; 94 samples were from females carrying a female fetus.

EXAMPLE 6 Redox Activity Assay with Polyaniline-Coated Film

Polyaniline-coated film was prepared by chemical deposition ofpolyaniline on a transparent film of poly(ethylene terephtahalate). 100ml of the reaction mixture comprised 1 M sulfuric acid, 1 ml of aniline,0.9 g of potassium iodate and 1 g of 5-sulfosalicyclic acid. U.S. Pat.No. 5,451,526. A deposition time of 2.5 hours at room temperature wasused to produce the polyaniline film. The polyaniline coatings on bothsides of the film were carefully washed to remove loose deposits. Thefilm was dried, cut into small pieces (3 mm×2.0 cm), and stored in aclean container before use. Immediately prior to use, the film wasequilibrated in an acidic ferric chloride solution for 2 minutes toconvert all the polyaniline to its emaraldine state, which is theoxidized state. 1.75 ml of urine was processed by passing through 160 mgof acidic alumina column, and transferred to a cuvette. ThePolyaniline-coated film was carefully washed with deionized water andtransferred to the cuvette containing aged or processed urine. Theoptical density of the liquid in the cuvette was read at wavelength 630nm on a spectrophotometer. Alternatively, after washing, the film wasdipped in the purified urine fraction for 1 second before being takenout for visual observation of its change in color. Under the mildcondition of this assay, the aged and processed urine from a femalecarrying a female fetus has a comparatively higher overall reducingactivity than urine from a female carrying a male fetus, and therefore,was able to reduce the emeraldine form of polyaniline (blue) to its morereduced leucoemeraldine state (off-white). An aged and processed urinesample from a female bearing a male fetus did not react with theemeraldine form of polyaniline (blue). As a result, the film remainedblue in color. When fresh urine samples were used without processing, nodifference could be observed in this test.

EXAMPLE 7 Pre-Conception Assay

The overall redox activity of the body fluid during a menstrual cyclewithin which a female conceives is similar to the redox activity of herbody fluid after conception.

Namely, if her pre-conception body fluid has a comparatively higheroverall reducing activity than that of the body fluid from anon-pregnant female carrying an ovum compatible with fertilization by anX chromosome-bearing sperm under the harsh condition (for example, inthe FRAP assay), her ovum is compatible with fertilization by a Ychromosome-bearing sperm. Consistently, her post-conception urine samplewould also have a higher overall reducing activity than that of urinefrom a female carrying a female fetus, which is correlated with a malefetus. Conversely, if her pre-conception urine sample has a higheroverall oxidizing activity than that of urine from a non-pregnant femalecarrying an ovum compatible with fertilization by a Y chromosome-bearingsperm under harsh conditions, her ovum is compatible with fertilizationby an X chromosome-bearing sperm. Likewise, her post-conception urinesample would also have a higher overall oxidizing activity than that ofurine from a female carrying a male fetus, which is correlated with afemale fetus.

The FRAP assay was used to test both pre-conception and post-conceptionurine samples obtained from the same customer. Pre-conception urinesamples were collected during ovulation period for at least twoconsecutive months. Post-conception urine samples were collected on thefirst or second day of missed menstruation. If the pre-conceptionsamples indicated an alternating ovulation pattern, then the customerwere advised as follows. If the customer desired a baby boy, she wasadvised to try conception in months when the ova being produced werecompatible with fertilization by a Y chromosome-bearing sperm. If thecustomer desired a baby girl, she was advised to try conception inmonths when the ova being produced were compatible with fertilization byan X chromosome-bearing sperm.

Those customers whose urine samples indicated ova being released in twoconsecutive months had the same gender specific compatibilities wereadvised to collect more urine samples for additional tests. However,customers of this category did not wish to incur more costs and,therefore, did not continue after the initial tests.

Table 3 tabulates the results of pre-conception tests we conducted for130 customers during the period between 2006 and August 2008.

TABLE 3 Number of customers who received the pre-conception test andlater the post-conception test = 130 Number of Number of Number ofNumber of customers who customers who customers who customers whodesired a baby boy confirmed having a desired a baby girl confirmedhaving a and tried conception baby boy = 52 and tried conception babygirl = 41 according to our (Few did not call according to our (Few didnot call method = 71 back*.) method = 59 back*.) Success rate forconception of Success rate for conception of baby baby boy = 73.2% girl= 69.5% Total success rate of pre-conception test = 71.3% *As a policy,we never call a customer for a feedback due to confidentiality concerns.Examples 8-12 illustrate various methods to process urine samples beforeassaying its overall redox activity.

EXAMPLE 8 Processing of Urine Samples with Acidic Alumina, CalciumChloride and Lead Acetate

The pH of 3 ml urine sample was adjusted to 6.4 with 1 M HCl. The urinesample was then centrifuged to remove insoluble components. Thesupernatant was loaded to a column containing 200 mg acidic alumina toremove interfering agents. 50ul of 10% Na₂HPO₄ and 80 mg of calciumchloride were then added to the cleared urine sample. The reactionmixture was incubated at room temperature for 5 minutes. 10 mg of leadacetate was added to the sample and the test tube was shaken until alllead acetate dissolved. After 5 minutes, the pH of the sample wasadjusted to 8.5 with 2.5 M NaOH. The sample was then centrifuged at12000 rpm for 2 minutes to remove insoluble components. The processedurine sample was then assayed using the PTA or FRAP assay (Examples 2and 3).

EXAMPLE 9 Processing of Urine Samples with Sulfuric Acid and Florisil

1 ml of fresh urine was added to a microcentrifuge tube and incubated onice. Concentrated sulfuric acid was slowly added to the urine sample toreach its final concentration of 3 M in the urine sample. Themicrocentrifuge tube was inverted slowly several times and the mixtureincubated at 37° C. for 24 hours. The sample was then centrifuged at12000 rpm for 2 minutes before the supernatant was passed throughFlorisil to result in processed urine sample. 1 Omg of crystallinephospho-24-tungstic acid was then added to 0.75 ml of this processedurine sample, and the PTA assay conducted (Example 2).

EXAMPLE 10 Processing of Urine Samples with Ion Exchange Resins

The pH of 2 ml of urine sample was adjusted to pH 5.3 with dilutedglacial acetic acid. The urine sample was then centrifuged at 12000 rpmfor 2 minutes. The supernatant was batch treated with 160 mg of wellwashed Dowex™ resins (Dow Chemical Company) 1X 8-100 mesh Cl⁻ form ionexchange resin to remove porphyrins. This cleared urine sample was thenprocessed as described in Example 8. The processed urine sample wasassayed using the PTA or FRAP assay (Examples 2 and 3).

EXAMPLE 11 Processing of Urine Samples with Nylon Filter

The pH of 2ml of urine sample was adjusted to 4.0 with 1 M HCl, beforethe urine sample was centrifuged at 12000 rpm for 2 minutes. Thesupernatant was passed through 0.2 um Nylon filter to remove porphyrins.This cleared urine sample was then processed as described in Example 8.

EXAMPLE 12 Processing of Urine Samples with Acidic Alumina and Talc

In order to develop a home based gender test, 2 ml of urine sample wasadded to 200 mg of acidic alumina and talc, and the tube was shaken toensure mixing. The sample was incubated at room temperature for 5minutes, before the sample was filtered to obtain processed urinesample. The processed urine sample was assayed using the FRAP assay(Example 3).

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

1. A method of determining the gender of an unborn child comprising thesteps of: (a) contacting a body fluid from a pregnant female with atleast one redox indicator; (b) measuring redox activity of the bodyfluid; and, (c) comparing the redox activity of the body fluid with atleast one standard to determine the gender of the unborn child.
 2. Themethod of claim 1, wherein the body fluid is selected from the groupconsisting of urine, tears, saliva, sweat, blood, plasma, serum,cerebrospinal fluid and amniotic fluid.
 3. The method of claim 2,wherein the body fluid is urine.
 4. The method of claim 1, wherein thebody fluid is obtained during a period ranging from about the first dayof a missed menstruation to about 40 weeks of pregnancy.
 5. The methodof claim 4, wherein the body fluid is obtained during a period rangingfrom about 5 weeks to about 15 weeks of pregnancy.
 6. The method ofclaim 1, wherein the redox indicator comprises a heteropoly acid or itssalts.
 7. The method of claim 6, wherein the heteropoly acid isphospho-24-tungstic acid.
 8. The method of claim 1, wherein the redoxindicator is a chromogenic chemical.
 9. The method of claim 8, whereinthe chromogenic chemical comprises an oxidation-reduction sensitivemetallic ion.
 10. The method of claim 9, wherein the metallic ion isselected from the group consisting of copper, iron, chromium, tungstenand molybdenum ions in an oxidized state.
 11. The method of claim 8,wherein the chromogenic chemical is selected from the group consistingof ferric tripyridyltriazine (Fe(III)-TPTZ) complex and potassiumferricyanide.
 12. The method of claim 1, wherein the redox activity ismeasured using a free radical.
 13. The method of claim 12, wherein thefree radical is a hydroxyl radical.
 14. The method of claim 12, whereinsodium salicylate is used as the redox indicator.
 15. The method ofclaim 1, wherein the redox activity is measured using an electrochemicalsensor.
 16. The method of claim 1, wherein the redox activity ismeasured on a solid substrate which comprises a redox indicator.
 17. Themethod of claim 16, wherein the redox indicator is a chromogenicchemical.
 18. The method of claim 17, wherein the chromogenic chemicalis impregnated on a strip.
 19. The method of claim 1, further comprisingthe step of processing the body fluid prior to step (a).
 20. The methodof claim 19, wherein the body fluid is urine.
 21. The method of claim19, wherein the body fluid is aged for at least about 1 week at roomtemperature ranging from about 20° C. to about 30° C. prior to step (a).22. The method of claim 21, wherein the body fluid is aged at roomtemperature for a period of time ranging from about 1 week to 52 weeksprior to step (a).
 23. The method of claim 19, wherein the processingcomprises chemical, biochemical, physical or biological means.
 24. Themethod of claim 23, wherein the processing comprises enzymatictreatment.
 25. The method of claim 23, wherein the processing comprisesextraction.
 26. The method of claim 23, wherein the processing comprisespurification.
 27. The method of claim 19, wherein the processingcomprises using adsorbants selected from the group consisting of talc,silica-based particles such as silica gel, alumina, florisil, charcoal,kaolin, concanavaline A and its conjugates, calcium phosphate, calciumhydroxide, calcium chloride, Cetyltrimethyl ammonium bromide, lectin,protein or glycoprotein hydrolyzing enzymes, glassfiber filter, ionexchange resins, affinity ligands, organic solvents, solid phaseextractants, size exclusion sieves, and reverse phase chromatographicmaterials.
 28. The method of claim 19, wherein the processing comprisesusing precipitants.
 29. The method of claim 28, wherein the precipitantscomprise heavy metals selected from the group consisting of barium,lead, molybdenum and tungsten.
 30. The method of claim 28, wherein theprecipitants are selected from the group consisting of barium chloride,barium hydroxide, zinc chloride, mercuric chloride, lead acetate,ammonium sulphate, dextran, acetonitrile, chloroform, sodium hydroxide,trichloroacetic acid, potassium iodate and their mixtures.
 31. A kitcomprising, (a) a urine collecting vial; (b) a solid substratecomprising a redox indicator, wherein the redox indicator comprises atleast one chromogenic chemical; (c) printed material instructing a womanto collect urine and contact the urine with the solid substrate; and (d)printed material instructing a woman to compare redox activity of theurine sample with at least one standard to determine the gender of theunborn child based on change in color in the chromogenic chemical. 32.The kit of claim 31, wherein the kit further comprises chemicals toprocess urine.
 33. The kit of claim 31, wherein the printed materialfurther instructing a woman to process the urine prior to contact theurine with the solid substrate.
 34. A method of pre-conception babygender planning comprising the steps of: (a) contacting a body fluidfrom a non-pregnant female with at least one redox indicator; (b)measuring redox activity of the body fluid; and (c) comparing the redoxactivity of the body fluid with at least one standard to determinegender specific compatibility of the ovum released in a menstrual cycle.35. The method of claim 34, wherein the body fluid is urine.
 36. Themethod of claim 34, wherein the body fluid is obtained near ovulation ormiddle of the menstrual cycle.
 37. The method of claim 34, furthercomprising the step of processing the body fluid prior to step (a).